Abstract
Tendon-to-bone multi-tissue transition exhibits a hierarchical and continuous gradient of matrix composition and alignment, allowing for efficient transmission of mechanical loading between tendon and bone. Upon injury, main problems associated with tendon-to-bone regeneration include disorganized matrix deposition, with a gradual loss of mineral content resulting in poor mechanical properties, limiting tissue integration and the formation of a graded interface. Therefore, we propose to assembly two types of continuous microfibres with distinct topological and compositional features tailored to guide cell alignment and matrix deposition while matching the mechanical requirements of the native tissue.
Wet-spinning was used to produce textured composite microfibres using different flow rates and two polymer blends to replicate the anisotropic architecture of tendon (PCL/Gelatin, 22/9%, w/v) and the isotropic organization together with mineral composition of bone (PCL/Gelatin/Hydroxyapatite, 22/9% w/v and 7.7% w/w HAp). Obtained microfibres morphology, chemical and mechanical properties were evaluated. Biological performance was studied using human adipose-derived stem cells (hASCs). Cytoskeleton alignment, nuclei elongation and matrix mineralization were evaluated. Textile techniques were used to create a 3D fibrous scaffold. Morphological features were analyzed by micro-CT.
PCL/Gelatin fibers produced at 1 mL/h extrusion rate exhibited the highest anisotropic alignment, in opposition to PCL/Gelatin/HAp fibers produced under the same condition. Micro-CT analysis of PCL/Gelatin/HAp fibers demonstrated variations within pore diameter and particles size between the different flow rates. Herein, PCL/Gelatin fibers induced a higher cytoskeleton alignment and nuclei elongation (p < 0.0001) in seeded hASCs. In contrast, significantly higher mineralization was found in PCL/Gelatin/HAp fibres (day 7, p < 0.04; day 14, p < 0.0001) as observed by alizarin red staining and quantification, suggesting the induction of an osteogenic-like phenotype. As proof of concept, textile techniques were used to assemble the two types of fibers and create a 3D scaffold presenting a continuous gradient in HAp content, as well as topological cues. After 14 days of culture with hASCs, a gradient of collagen deposition and matrix mineralization was found (p < 0.014, p < 0.0001). Higher deposition of collagen type II was observed in the tendon and interface parts of the fibrous scaffold and collagen type X in the interface.
Overall, the wet-spinning method was efficiently used to engineer continuous textured composite microfibers. PCL/Gelatin fibers supported cell alignment mimicking tendon one, while PCL/Gelatin/HAp fibers induced mineral deposition and a possible phenotypic change without additional medium supplementation. Textile techniques allowed fibres assemblage and 3D scaffolds fabrication envisioning tendon-to-bone applications.
